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Sensory neurons in DRG express Robo1 and Robo2 after sciatic nerve transection injury, as detected by immunohistochemistry. (A-C) Double staining for Robo1 (red, Alexa Fluor 568) and NeuN (green, Alexa Fluor 488) showing Robo1 immunopositivity in the cell bodies of adult sensory neurons. (D-F) Double staining Robo2 (red, Alexa Fluor 568) and NeuN (green, Alexa Fluor 488) showing Robo2 expression in the cell bodies of adult sensory neurons. (G-I) Double staining Robo1 (red, Alexa Fluor 568) and NeuN (green, Alexa Fluor 488) showing Robo1 immunopositivity in the cell bodies of sensory neurons in L4 DRG at 7 days after sciatic nerve transection injury. (J-L) Double staining Robo2 (red, Alexa Fluor 568) and NeuN (green, Alexa Fluor 488) showing Robo2 immunopositivity in the cell bodies of sensory neurons in L4 DRG at 7 days after sciatic nerve transection injury. (M-N) In situ hybridization indicated Robo1 and Robo2 mRNA expression in the cell bodies of sensory neurons in L4 DRG at 7 days after sciatic nerve transection injury. Scale bars: 40 μm. DRG: Dorsal root ganglion; Robo: Roundabout.
Source publication
The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors. Although we previously reported that Slit1-3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,...
Contexts in source publication
Context 1
... staining with the neuronal marker NeuN confirmed that motor neurons in the ventral horn of the spinal cord express both Robo1 and Robo2 receptors after sciatic nerve transection injury (Figure 3C-H). Double staining of Robo1 and Robo2 with NeuN in DRG (L4-5) sections from the uninjured side revealed that Robo1 and Robo2 are expressed in sensory neurons before sciatic nerve transection injury (Figure 4A-F). Moreover, double staining Robo1 and Robo2 with NeuN in DRG (L4-5) sections from the injured side revealed that Robo1 and Robo2 are also expressed in sensory neurons after sciatic nerve transection injury (Figure 4G-L). ...
Context 2
... staining of Robo1 and Robo2 with NeuN in DRG (L4-5) sections from the uninjured side revealed that Robo1 and Robo2 are expressed in sensory neurons before sciatic nerve transection injury (Figure 4A-F). Moreover, double staining Robo1 and Robo2 with NeuN in DRG (L4-5) sections from the injured side revealed that Robo1 and Robo2 are also expressed in sensory neurons after sciatic nerve transection injury (Figure 4G-L). Because the NeuN antibody showed both nucleus and cytoplasmic staining, so, we further used in situ hybridization of DRG sections to detect Robo1 and Robo2 mRNA expression. ...
Context 3
... the NeuN antibody showed both nucleus and cytoplasmic staining, so, we further used in situ hybridization of DRG sections to detect Robo1 and Robo2 mRNA expression. The results not only showed Robo1 and Robo2 expression in sensory neurons, but also confirmed the specificity of the Robo1 and Robo2 antibodies (Figure 4M & N). Thus, Robo1 and Robo2 receptors are expressed in both motor and sensory neurons during peripheral axon regeneration. ...
Citations
... 55 In terms of neural repair, recent studies have shown that SLIT3 plays a crucial role in controlling the formation of new neural bridges and proper axon regeneration after peripheral nerve transection injuries, 56 as well as in peripheral nerve repair after injury. 57 The cornea is the most densely innervated tissue of the body; after corneal injury, the regeneration of the local sensory nerves is regulated by numerous complex molecules. 19,20 Different AGMs have distinct effects on corneal nerve growth. ...
Purpose:
In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration.
Methods:
Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells.
Results:
Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment.
Conclusions:
ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.
... Of particular interest were the Slit/Robo signalling pathways, whose repulsive signals 246 are known to play a role in axonal guidance (Brose et al., 1999;Jia et al., 2005;Kaneko et al., 247 2010;Kidd et al., 1999;Nguyen-Ba-Charvet et al., 2004;Ypsilanti et al., 2010). Previous studies 248 have shown that of the three Slit genes, Slit2 and Slit3 are predominately expressed by SCs (Carr 249 et al., 2017;Chen et al., 2020;Wang et al., 2013). We first confirmed this finding ( ...
Collective cell migration is fundamental for the development of organisms and in the adult, for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during rat Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell-surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective Schwann cell migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased Schwann cell collective migration and increased clustering of Schwann cells within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.
... Cellular reorganization after injury in the DRG has attracted the attention of researchers in recent years, and these have focused on cellular and molecular interactions in glial and sensory cells in the DRG following peripheral nerve injury (Chen et al. 2020;Dubovy et al. 2013;Matsumura et al. 2014;Muratori et al. 2015). In addition to such research, there are also stereological and immunohistochemical studies investigating the time-dependent change in cell death and new cell formation in the DRG following peripheral nerve axotomy (McKay Hart et al. 2002;Muratori et al. 2015;Schmalbruch 1987;Shi et al. 2001;Tandrup and Braendgaard 1994). ...
Peripheral nerve injuries lead to significant changes in the dorsal root ganglia, where the cell bodies of the damaged axons are located. The sensory neurons and the surrounding satellite cells rearrange the composition of the intracellular organelles to enhance their plasticity for adaptation to changing conditions and response to injury. Meanwhile, satellite cells acquire phagocytic properties and work with macrophages to eliminate degenerated neurons. These structural and functional changes are not identical in all injury types. Understanding the cellular response, which varies according to the type of injury involved, is essential in determining the optimal method of treatment. In this research, we investigated the numerical and morphological changes in primary sensory neurons and satellite cells in the dorsal root ganglion 30 days following chronic compression, crush, and transection injuries using stereology, high-resolution light microscopy, immunohistochemistry, and behavioral analysis techniques. Electron microscopic methods were employed to evaluate fine structural alterations in cells. Stereological evaluations revealed no statistically significant difference in terms of mean sensory neuron numbers (p > 0.05), although a significant decrease was observed in sensory neuron volumes in the transection and crush injury groups (p < 0.05). Active caspase-3 immunopositivity increased in the injury groups compared to the sham group (p < 0.05). While crush injury led to desensitization, chronic compression injury caused thermal hyperalgesia. Macrophage infiltrations were observed in all injury types. Electron microscopic results revealed that the chromatolysis response was triggered in the sensory neuron bodies from the transection injury group. An increase in organelle density was observed in the perikaryon of sensory neurons after crush-type injury. This indicates the presence of a more active regeneration process in crush-type injury than in other types. The effect of chronic compression injury is more devastating than that of crush-type injury, and the edema caused by compression significantly inhibits the regeneration process.
... The Robo family has four members that mediate a variety of neuronal responses, including neurogenesis, migration, branching, dendritic pattern formation, synaptogenesis, and axon guidance, 5 which play an important role in angiogenesis and tumorigenesis 6 and are involved in a variety of neurological diseases such as ischemic cerebrovascular disease 7 and peripheral neuropathy. 8 Robo1, a member of the Robo family, is widely expressed in the central nervous system. 9 Slit is a member of the Slit family of neural guidance factors (Slit1-3) and Slit/Robo signaling is first identified in the nervous system. ...
The neural network hypothesis is one of the important pathogenesis of drug‐resistant epilepsy. Axons guide molecules through synaptic remodeling and brain tissue remodeling, which may result in the formation of abnormal neural networks. Therefore, axon guidance plays a crucial role in disease progression. However, although Robo1 is one of the important components of axon guidance, the role of Robo1 in epilepsy remains unclear. In this study, we aimed to explore the mechanism of Robo1 in epilepsy. Male adult C57BL/6 mice were intraperitoneally injected with pentylenetetrazol to establish an epilepsy model. Lentivirus (LV) was given via intracranial injection 2 weeks before pentylenetetrazol injection. Different expressions of Robo1 between the control group, LV‐mediated Robo1 short hairpin RNA group, empty vector control LV group, and normal saline group were analyzed using Western blot, immunofluorescence staining, Golgi staining, and video monitoring. Robo1 was increased in the hippocampus in the pentylenetetrazol‐induced epilepsy mouse model; lentiviral Robo1 knockdown prolonged the latency of seizure and reduced the seizure grade in mice and resulted in a decrease in dendritic spine density, while the number of mature dendritic spines was maintained. We speculate that Robo1 has been implicated in the development and progression of epilepsy through its effects on dendritic spine morphology and density. Epileptic mice with Robo1 knockdown virus intervention had lower seizure grade and longer latency. Follow‐up findings suggest that Robo1 may modulate seizures by affecting dendritic spine density and morphology. Downregulation of Robo1 may negatively regulate epileptogenesis by decreasing the density of dendritic spines and maintaining a greater number of mature dendritic spines.
... In addition, glial cells in the lesioned area and other cells in the immune system also constitute a component of this response (Jager et al. 2020; Lu and Richardson 1993). Cellular reorganization after injury in the DRG has attracted the attention of researchers in recent years, and these have focused on cellular and molecular interactions in glial and sensory cells in the DRG following peripheral nerve injury (Chen et al. 2020;Dubovy et al. 2013; Matsumura et al. 2014; Muratori et al. 2015). In addition to such research, there are also stereological and immunohistochemical studies investigating the time-dependent change in cell death and new cell formation in the DRG following peripheral nerve axotomy (McKay Hart et al. 2002;Muratori et al. 2015;Schmalbruch 1987;Shi et al. 2001; Tandrup and Braendgaard 1994). ...
Peripheral nerve injuries lead to significant changes in the dorsal root ganglia, where the cell bodies of the damaged axons are located. The sensory neurons and the surrounding satellite cells rearrange the composition of the intracellular organelles to enhance their plasticity for adaptation to changing conditions and responding to injury. Meanwhile, satellite cells acquire phagocytic properties and work with macrophages to eliminate degenerated neurons. These structural and functional changes are not identical in all injury types. Understanding the cellular response, which varies according to the type of injury involved, is essential in determining the optimal method of treatment. In this research, we investigated the numerical and morphological changes in primary sensory neurons and satellite cells in the dorsal root ganglion 30 days following chronic compression, crush and transection injuries using stereology, high-resolution light microscopy, immunohistochemistry, and behavioral analysis techniques. Electron microscopic methods were employed to evaluate fine structural alterations in cells. Stereological evaluations revealed no statistically significant difference in terms of mean sensory neuron numbers (p > 0.05), although a significant decrease was observed in sensory neuron volumes in the transection and crush injury groups (p < 0.05). Active caspase-3 immunopositivity increased in the injury groups compared to the sham group (p < 0.05). While crush injury led to desensitization, chronic compression injury caused thermal hyperalgesia. Electromyography parameters exhibited a significant decrease in the compression and crush injury groups compared to the sham group (p < 0.05). Macrophage infiltrations were observed in all injury types. Electron microscopic results revealed that the chromatolysis response was triggered in the sensory neuron bodies from the transection injury group. An increase in organelle density was observed in the perikaryon of sensory neurons after crush-type injury. This indicates the presence of a more active regeneration process in crush-type injury than in other types. The effect of chronic compression injury is more devastating than that of crush-type injury, and the edema caused by compression significantly inhibits the regeneration process.
... Our data showed that neuron stage A was enriched in genes involved in the pan cell cycle markers Mcm6 and Mcm5, suggesting that this stage may be a transitional state from progenitor cells to neurons (Pacal and Bremner 2012). The axon development gene Robo2 was specifically enriched in neuron stage B (López-Bendito et al. 2007;Zhang et al. 2012;Chen et al. 2020). Neuron stage C expressed Foxp2, Foxp4, and synaptotagmin-1 (Syt1). ...
... In the current study, we found that Cd interfered with neuron development by reducing the number of new neurons and decreasing the complexity of neuronal axons and dendrites. Pseudotime analyses further revealed that Cd altered the trajectory of maturation of neuronal lineage cells by hindering the maturation of cells, especially the low maturity neuron stage B. Notably, Robo2 expression was significantly enriched in neuron stage B, and Robo2-Slit2 signaling has been shown to play an important role in early axonal development (López-Bendito et al. 2007;Zhang et al. 2012;Chen et al. 2020). These results indicated that neurites have begun to sprout at the stage of neuron B, while Cd exposure greatly affects this critical period of neuron development. ...
Cadmium (Cd) is an extensively existing environmental pollutant that has neurotoxic effects. However, the molecular mechanism of Cd on neuronal maturation is unveiled. Single-cell RNA sequencing (scRNA-seq) has been widely used to uncover cellular heterogeneity and is a powerful tool to reconstruct the developmental trajectory of neurons. In this study, neural stem cells (NSCs) from subventricular zone (SVZ) of newborn mice were treated with CdCl2 for 24 h and differentiated for 7 days to obtain neuronal lineage cells. Then scRNA-seq analysis identified five cell stages with different maturity in neuronal lineage cells. Our findings revealed that Cd altered the trajectory of maturation of neuronal lineage cells by decreasing the number of cells in different stages and hindering their maturation. Cd induced differential transcriptome expression in different cell subpopulations in a stage-specific manner. Specifically, Cd induced oxidative damage and changed the proportion of cell cycle phases in the early stage of neuronal development. Furthermore, the autocrine and paracrine signals of Wnt5a were downregulated in the low mature neurons in response to Cd. Importantly, activation of Wnt5a effectively rescued the number of neurons and promoted their maturation. Taken together, the findings of this study provide new and comprehensive insights into the adverse effect of Cd on neuronal maturation.
Graphical abstract
... GAP-43 participates in regulating axonal elongation, and SYN is beneficial to axonal sprouting and synaptogenesis (Chung et al., 2020;Jing et al., 2020). In particular, the coordinated action of attractive and repulsive extracellular axonal guidance molecules, including Netrins and DCC, Slit-2, and Robo-1, is important for neurite growth and guidance (Chen et al., 2020;Cuesta et al., 2020). In addition to neurite growth and guidance, axonal regeneration could be constrained by neurite growth inhibitors. ...
Ischemic stroke elicits white matter injury typically signed by axonal disintegration and demyelination; thus, the development of white matter reorganization is needed. 2,3,5,6-Tetramethylpyrazine (TMP) is widely used to treat ischemic stroke. This study was aimed to investigate whether TMP could protect the white matter and promote axonal repair after cerebral ischemia. Male Sprague–Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and treated with TMP (10, 20, 40 mg/kg) intraperitoneally for 14 days. The motor function related to gait was evaluated by the gait analysis system. Multiparametric magnetic resonance imaging (MRI) was conducted to noninvasively identify gray-white matter structural integrity, axonal reorganization, and cerebral blood flow (CBF), followed by histological analysis. The expressions of axonal growth-associated protein 43 (GAP-43), synaptophysin (SYN), axonal growth-inhibitory signals, and guidance factors were measured by Western blot. Our results showed TMP reduced infarct volume, relieved gray-white matter damage, promoted axonal remodeling, and restored CBF along the peri-infarct cortex, external capsule, and internal capsule. These MRI findings were confirmed by histopathological data. Moreover, motor function, especially gait impairment, was improved by TMP treatment. Notably, TMP upregulated GAP-43 and SYN and enhanced axonal guidance cues such as Netrin-1/DCC and Slit-2/Robo-1 but downregulated intrinsic growth-inhibitory signals NogoA/NgR/RhoA/ROCK-2. Taken together, our data indicated that TMP facilitated poststroke axonal remodeling and motor functional recovery. Moreover, our findings suggested that TMP restored local CBF, augmented guidance cues, and restrained intrinsic growth-inhibitory signals, all of which might improve the intracerebral microenvironment of ischemic areas and then benefit white matter remodeling.
... SLITs are the secreted protein and ROBOs are their receptors. An experiment of sciatic nerve crush in mice revealed the downregulation of Slit2 after injury, but also its upregulation during axonal regeneration, which could be the case of our comparison where Slit2 is slightly upregulated [63]. The downregulation of the repellent signal from Slit3 may be interpreted as the Unc5b one, so the regeneration has just started and there is no point to repel an axon that is not already grown. ...
Spinal cord injury affects the lives of millions of people around the world, often causing disability and, in unfortunate circumstances, death. Rehabilitation can partly improve outcomes and only a small percentage of patients, typically the least injured, can hope to return to normal living conditions. Cannabis sativa is gaining more and more interest in recent years, even though its beneficial properties have been known for thousands of years. Cannabigerol (CBG), extracted from C. sativa, is defined as the “mother of all cannabinoids” and its properties range from anti-inflammatory to antioxidant and neuroprotection. Using NSC-34 cells to model spinal cord injury in vitro, our work evaluated the properties of CBG treatments in motor neuron regeneration. While pre-treatment can modulate oxidative stress and increase antioxidant enzyme genes, such as Tnx1, decreasing Nos1 post-treatment seems to induce regeneration genes by triggering different pathways, such as Gap43 via p53 acetylation by Ep300 and Ddit3 and Xbp1 via Bdnf signaling, along with cytoskeletal remodeling signaling genes Nrp1 and Map1b. Our results indicate CBG as a phytocompound worth further investigation in the field of neuronal regeneration.
... In recent years, an increasing number of studies have found that axon guiding molecules such as Sema [17], Netrins [18,19], and Slit [20,21] are also Ivyspring International Publisher members of the neuroskeletal network. They play an essential role in neural development and are expressed in the bone microenvironment to mediate the interaction between osteoblasts and osteoclasts. ...
Slit/Robo signals were initially found to play an essential role in nerve development as axonal guidance molecules. In recent years, with in-depth study, the role of Slit/Robo in other life activities, such as tumor development, angiogenesis, cell migration, and bone homeostasis, has gradually been revealed. Bone is an organ with an active metabolism. Bone resorption and bone formation are closely related through precise spatiotemporal coordination. There is much evidence that slit, as a new bone coupling factor, can regulate bone formation and resorption. For example, Slit3 can promote bone formation and inhibit bone resorption through Robo receptors, which has excellent therapeutic potential in metabolic bone diseases. Although the conclusions of some studies are contradictory, they all affirm the vital role of Slit/Robo signaling in regulating bone metabolism. This paper reviews the research progress of Slit/Robo signaling in bone metabolism, briefly discusses the contradictions in the existing research, and puts forward the research direction of Slit/Robo in the field of bone metabolism in the future.
... Establishment of the sciatic nerve crushing (SNC) and SNT model. In order to evaluate the neurovascular regenerative ability of the Pc-NVs, the SNc and SNT model were prepared as described previously (21,22). The SNc model was used for the tracking analysis of the injected Pc-NVs. ...
Pericyte‑derived extracellular vesicle‑mimetic nanovesicles (PC‑NVs) play an important role in the improvement of erectile function after cavernous nerve injury. However, the impact of PC‑NVs on the peripheral nervous system (PNS), such as the sciatic nerve, is unclear. In this study, PC‑NVs were isolated from mouse cavernous pericytes (MCPs). A sciatic nerve transection (SNT) model was established using 8‑week‑old C57BL/6J mice. The sciatic nerve was harvested 5 and 14 days for immunofluorescence and western blot studies. Function studies were evaluated by performing the rotarod test and walking track analysis. The results demonstrated that PC‑NVs could stimulate endothelial cells, increase neuronal cell content, and increase macrophage and Schwann cell presence at the proximal stump rather than the distal stump in the SNT model, thereby improving angiogenesis and nerve regeneration in the early stage of sciatic nerve regeneration. In addition, PC‑NVs also increased the expression of neurotrophic factors (brain‑derived nerve growth factor, neurotrophin‑3 and nerve growth factor) and the activity of the cell survival signaling pathway (PI3K/Akt signaling), and reduced the activity of the JNK signaling pathway. Additionally, after 8 weeks of local application of PC‑NVs in SNT model mice, their motor and sensory functions were significantly improved, as assessed by performing the rotarod test and walking track analysis. In conclusion, the present study showed that the significant improvement of neurovascular regeneration in mice following treatment with PC‑NVs may provide a favorable strategy for promoting motor and sensory regeneration and functional recovery of the PNS.
